Monitoring of Proteus distribution by environmental DNA sampling


 eDNA Proteus

(Photo: Gregor Aljančič)


Tular Cave Laboratory (Society for Cave Biology) & partners developed a research tool to overcome the critical problem when determining Proteus occurrence – inaccessibility of its subterranean habitat. This highly efficient, non-invasive and innovative molecular genetic tool helps to detect traces of Proteus environmental DNA (eDNA) by filtering water samples from groundwater (karst springs, wells or caves). Coupled with GIS analysis, the method was implemented to test for the presence of Proteus in new potential localities, regardless of whether or not the animal has been observed there, as well as to verify selected old data on its distribution.

The results of this study offered a complete picture of Proteus distribution within the study area and its most vulnerable parts. Findings were published on 27th March2017 in the journal Scientific Reports: "Environmental DNA in subterranean biology: range extension and taxonomic implications for Proteus" (Read open access article).


Main results of the study (read more in PR announcement)

  • The most extensive survey of Proteus distribution ever conducted: 56 karst springs, wells or caves in Slovenia, Bosnia & Herzegovina and Montenegro were sampled and analysed (95 visited).
  • A likely presence of Proteus confirmed at seven new sites along the southern limit of its known range.
  • First significant extension of Proteus range after 1929: the presence of Proteus documented for the first time in Montenegro.
  • A highly specific method was developed to detect the eDNA of the black Proteus morph, which occurs only on 30 km2 in Southeastern Slovenia.
  • A major increase in the number of known black Proteus sites since its discovery in 1986: the black Proteus was detected at five new sites, which more than doubled the number of sites of this extremely vulnerable population – basis for an efficient conservation management.
  • The eDNA of both black and white Proteus was discovered together in one of the springs, which represents the first evidence that these two populations may be in contact with each other. 


The study was organized and conducted by the Tular Cave Laboratory (Society for Cave Biology, Slovenia), in partnership with the Department of Animal Science (Biotechnical Faculty, University of Ljubljana, Slovenia), the Evolutionary Zoology Laboratory (Institute of Biology at the Scientific Research Centre of the Slovenian Academy of Sciences and Arts, Slovenia), the Jeffery Lab (Department of Biology, University of Maryland, USA) and the Department of Life Sciences (University of Trieste, Italy), field work was supported by the Biospeleological Society of Montenegro, the Scientific Research Society Versus (Bosnia & Herzegovina) and Center for Karst and Speleology (Bosnia & Herzegovina).

The study was part of the project “A survey of the distribution of Proteus anguinus by environmental DNA sampling”, co-financed by the Critical Ecosystem Partnership Fund, BirdLife International and DOPPS (CEPF GEM No. 45), and the project “With Proteus we share dependence on groundwater”, financed by the EEA Financial Mechanism and the Norwegian Financial Mechanism 2009-2014 (SI03-EEA2013/MP-17).


Program of the International workshop “Conservation of cave biodiversity in Southeast Dinaric Karst” (Shkodër, Albania, 26. 10. 2016; PDF: 0,49KB)


 Rijeka Crnojevica MNE Large   Sopot Cave MNELarge    Trebizat CEPF BE Large    Hutovo Blato photo Gregor Aljancic a  
 Bela Krajina SLO Large    Bregava BE Large    Info Board Vitina BE Large   Hutovo Blato BE Large   
eDNA SLO Large   eDNA Proteus 2   eDNA1 SLO Large   Filtering  


The study was part of projects:

 I. "Monitoring of Proteus anguinus by environmental DNA sampling" (2013-2014)

Tular logo Rodica M cepf PARTNERJI1                                                    cepf logos1

Project was cofinanced by Critical Ecosystem Partnership Fund, BirdLife International and DOPPS, 2013–2014

Final project completion report (PDF, 3.89MB)


Finding and protecting hidden species: environmental DNA

Amongst cold, dark caves of dripping stalagtites, there is a diverse community of highly-adapted subterranean species. A scientific breakthrough is helping ensure they are monitored and protected in the future. (Read more)

CEPF Top articles: Cave-Dwelling Salamander Sheds Light on Drinking Water Resources

BirdLife International: Scientific breakthrough reveals evidence of ‘human fish’ locked away in cave system

Conserving the Biodiversity of the Balkans: A Long-Term Strategic Vision for Europe’s natural jewel

Proteus is the flagship species of the Dinaric karst groundwater and its endangered cave fauna. As such, Proteus represents our joint aspiration to raise scientific and public attention needed to preserve them in the future!

Take a visual journey through the Mediterranean Basin Biodiversity Hotspot - explore its StoryMap!

"This network is not just 93 civil society organisations supported by CEPF. It is also 93 organisations who work together more and more as a team to preserve the amazing biodiversity of this region. Despite facing a lot of difficulties today and in the future, civil society organisations are finding inspiration in the work of their fellow CEPF partners from other countries in the hotspot." , said Pierre Carret, Grant Director, CEPF (read more).



II. With Proteus we share dependence on groundwater" (2015)

Tular logo   Dobrepolje1                               

Applicant: Society for Cave Biology
Partners: Institute of Biology ZRC SAZU, Municipality of Črnomelj and Municipality of Dobrepolje, in co-operation with Department of Animal Sciences, Biotechnical Faculty, University of Ljubljana

The project is supported by the EEA Financial Mechanism and the Norwegian Financial Mechanism 2009-2014. 

Project's summary









 eDNA Proteus 2     NO3 Bela krajina Workshop SOS Proteus 2015

New forensic method developed to detect molecular traces of rare black Proteus in groundwater (environmental DNA).

Number of localities of highly endangered black Proteus doubled.

Updated distribution map of black Proteus.

GIS model indicating the current pollution threats on Proteus subterranean cave habitat and groundwater.

Discussion on implementation of monitoring and practical actions for conservation of Proteus in Slovenia. New methods and solutions shared (Dobrepolje, Slovenia, 12/12/2015).

Program of SOS Proteus (PDF 1.4MB)







Threats on Proteus and groundwater Page 1s  lecture Dragatus Zatocisce za cloveske ribice Page 1s
Conservation of Proteus and other endemic cave animals is not merely an attempt to preserve the subterranean biodiversity, with cave animals we share the most important resource of fresh water – groundwater. Conservation of Proteus and its subterranean habitat may truly be effective only by public outreach, emphasizing vulnerability of karst landscape.

Sanctuary for Proteus accidentally washed‐out of their subterranean habitat during seasonal flooding.

After veterinary inspection and care animals are retleased their source populations.

Leaflet (PDF; 1.3MB) Leaflet, in Slovene (PDF; 1.6MB)



Short documentary film SOS Proteus

film on conservation of Proteus and protection of groundwater in Slovenia (English version in preparation)

The film consists of three parts:

1. The idyllic Karst and the myth of Proteus, 2. the reality of pollution in the karst underground, and, 3. threats on the black Proteus and drinking water in Bela Krajina, SE Slovenia.



>READ MORE: Vulnerability of Proteus and groundwater